C.J.H. Kelnar

To study the ontogeny of spontaneous pulsatile LH and FSH secretion before the onset of puberty, plasma LH and FSH were measured by an ultrasensitive time-resolved immunoflurometric assay in 16 boys and 6 girls, aged 6.5 +/- 0.2 yr (+/- SEM; range, 4.4-8.0) with short stature. Eight male patients with idiopathic hypogonadotropic hypogonadism (Kallmann's syndrome), aged 24.1 +/- 3.4 yr, were also investigated. Blood samples were withdrawn at 10- to 20-min intervals for 12 h from 2000-0800 h. Pituitary responsiveness was assessed by a standard iv LHRH challenge test. LH and/or FSH pulses were detectable in all but two prepubertal subjects. In boys, low amplitude LH (0.16 +/- 0.06 U/L) and FSH (0.19 +/- 0.03 U/L) pulses were detectable at mean frequencies of 2.19 +/- 0.37 and 2.13 +/- 0.46 pulses/12 h, respectively. In girls, low amplitude LH (0.29 +/- 0.18 U/L) pulses, but higher (P less than 0.05 compared to boys) amplitude FSH (1.62 +/- 1.05 U/L) pulses were observed at frequencies of 1.71 +/- 0.56 and 1.67 +/- 0.53 pulses/12 h, respectively. Mean FSH in prepubertal girls (1.95 +/- 0.88 U/L) was significantly (P less than 0.05) higher than that in boys (0.46 +/- 0.07 U/L), but mean LH was not different at 0.17 +/- 0.07 and 0.10 +/- 0.03 U/L, respectively. Patients with Kallmann's syndrome had mean LH and FSH levels indistinguishable from those of prepubertal boys. Nocturnal augmentation of pulsatile LH or FSH secretion was observed in 74% of children (71% in girls and 75% in boys), but in none of the eight patients with Kallmann's syndrome. A close temporal association was observed between sleep onset and the appearance of nocturnal pulsatile gonadotropin secretion. The FSH response to exogenous LHRH in prepubertal girls was significantly greater than that in patients with Kallmann's syndrome and prepubertal boys, but LH responses were not different. Our results show that pulsatile LH and FSH secretion occurs in the majority of boys and girls in midchildhood, with a robust association with nocturnal sleep onset. Between the ages of 4-8 yr, these low amplitude and low frequency pulses are unable to activate gonadal function. The regulation of FSH secretion in prepubertal girls appears to be different from that in prepubertal boys.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND: Testicular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans. METHODS: Immunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2gamma, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat. RESULTS: In marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period. CONCLUSIONS: The marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

The ontogeny of gonadotropin releasing hormone pulse generator activity underlying pubertal development in the human male is incompletely defined because of the limitations of assay sensitivity in measurements and the inaccuracies attendant upon the analyses of pulsatile secretion of circulating gonadotropins. Using an ultrasensitive immunofluorometric assay (DELFIA) to measure plasma LH and deconvolution analysis to depict LH secretory characteristics, we compared nocturnal (2000-0800 h) pulsatile LH secretion cross-sectionally in 16 boys in midchildhood (mean +/- SD age 6.6 +/- 0.3 yr), 8 prepubertal boys (12.0 +/- 0.3 yr), 8 early pubertal boys (14.3 +/- 0.4 yr), and in 8 young fertile adult men (32.6 +/- 1.6 yr) as an indirect in vivo assessment of hypothalamic GnRH pulse generator activity over the entire span of pubertal development in the human male. We confirmed that sleep-entrained GnRH/LH burst secretory activity was present in midchildhood. The first increase in sleep-entrained GnRH/LH secretion occurred some 2 yr before the clinical onset of puberty. From midchildhood to sexual maturity, LH production rate increased 39-fold. However, GnRH/LH pulse frequency showed only a relatively small (1.8-fold) increment from midchildhood to the clinical onset of puberty, with no subsequent changes to continuing development towards adulthood. Thus 91.7% of the increment in LH plasma concentration from childhood to sexual maturity could be accounted for by an amplification of a pre-existing ultradian rhythm of secretion with a steadily and markedly increasing mass of LH secreted per burst. The duration of secretory burst and apparent half-life of plasma LH disappearance remained constant from midchildhood, through puberty, to adulthood. The nyctohemeral rhythm-and sleep-associated LH/GnRH secretion was eventually lost in young adulthood. We conclude that the onset of puberty in man is heralded by the reawakening of a partially quiescent GnRH pulse generator. This predominantly involves an amplification of a pre-existing pattern of hypothalamic GnRH secretion leading to a major augmentation of the total quantity of LH molecules released per burst. The almost two-fold increment in GnRH pulse frequency contributed synergistically to the pubertal process, before the clinical onset of puberty, possibly by enhancing gonadotropic sensitivity to increase the mass of LH produced per burst. The relative constancy of GnRH pulse frequency in the gonad-intact hypothalamic-pituitary-testicular axis from pubertal onset to adulthood implies that testicular steroidal feedback plays a role in restraining the burst frequency of the GnRH pulse generator during pubertal development and adulthood.

BACKGROUND: Inexplicably, boys treated with some therapies for cancer at age 2-10 years, a time of supposed 'testicular quiescence', are at risk of low sperm counts/infertility in adulthood. Our aims were to use the marmoset as a surrogate for man to establish testicular cell function/activity during 'quiescence' between the neonatal period and puberty, and to test if any cell activity could be suppressed by prior treatment with a GnRH antagonist. METHODS AND RESULTS: Based on immunoexpression studies, functional development of Sertoli cells (SGP-2, androgen receptor) and Leydig cells (3 beta-hydroxysteroid dehydrogenase) was detectable at an age (35 weeks) when the testis is considered to be quiescent, and in advance of the pubertal rise in blood testosterone levels (50-60 weeks). Other changes at 35 weeks were the appearance of focal seminiferous tubule lumens and proliferating germ cells [indicated by immunoexpression of proliferating cell nuclear antigen (PCNA)]. Treatment from 25 to 35 weeks with GnRH antagonist largely (>85%) prevented these changes. However, the PCNA-labelling index of spermatogonia in GnRH antagonist-treated animals did not differ from controls (41.3 versus 43.6%) though total spermatogonia volume per testis was reduced by 41%. Some protein markers (inhibin-alpha, estrogen receptor-beta) showed little change with age or treatment. Beyond 35 weeks, GnRH antagonist-treated animals showed a delay in the pubertal rise in plasma testosterone levels. CONCLUSIONS: These findings reinforce the view that the 'childhood' testis is not quiescent. This may explain the damaging effects of some cancer therapies on subsequent fertility of boys and raises the issue of protective intervention. The present studies suggest that GnRH antagonist-based intervention might be only partially successful. Identification of the factors regulating spermatogonial development in the infant marmoset may aid in the design of such strategies.